All references, including patents, patent applications, and non-patent publications, cited throughout are hereby expressly incorporated by reference in their entireties for all purposes.
Antibody display technologies, such as phage and yeast display, have proven powerful alternatives to natural antibodies for generating high affinity antibodies for therapeutics and diagnostics (Bradbury et al., 2011, Feldhaus et al., 2003, Fuh, 2007, Hanes et al., 2000, Hoogenboom, 2005, Winter et al., 1994). There are several advantages of in vitro technologies compared to traditional hybridoma systems, including the ability to simultaneously screen very large numbers of antibodies for binding to antigens of interest, overcome immunological tolerance, and further mature selected antibodies to higher affinities than can be achieved in vivo (see, e.g., Boder et al., 2000, Boder et al., 1997, Bostrom et al., 2009, Bostrom et al., 2009, Garcia-Rodriguez et al., 2011, Hanes, et al., 2000, and Lou et al., 2010). One of the more significant advantages of antibody library technology is the ability to control selection conditions to preferentially select for binders with desirable properties. For instance, a number of methods have been employed in order to enrich for high affinity clones, such as lowered antigen concentrations, increased washing stringency, and the inclusion of unlabeled antigen to pressure clones with fast dissociation rates (see, e.g., Boder, et al., 2000, Bostrom, et al., 2009, Hawkins et al., 1992, and Winter, et al., 1994). Selections can also be performed at high temperatures or in the presence of chaotropic agents to enrich for clones with high thermostability (see, e.g., Pavoor et al., 2012, and Traxlmayr et al., 2013). In addition, cross-reactive clones can be recovered by performing sequential selections with related antigens (see, e.g., Bostrom, et al., 2009, Fagete et al., 2012, Garcia-Rodriguez, et al., 2011, Schaefer et al., 2011).
One of the more definitive feature of an antibody is its epitope(s), which stereospecifically determine(s) any functional activity of the antibody/antigen complex (see, e.g., Deng et al., 2013, Ekiert et al., 2012, Felding-Habermann et al., 2004, Kong et al., 2012, Kwong et al., 2012, and Kwong et al., 2009). When available, lead antibodies or ligands that target a desired epitope allows these reagents to be used as competitors during selections to enrich for clones of cross-blocking specificity. However, as is often the case such control reagents are unavailable and/or the identity, nature, and/or number of functional epitopes on an antigen of interest is not known, it is desirable—indeed, often necessary—to isolate and characterize antibodies against a broad range of different epitopes in order to screen for functional clones. Unfortunately, however, many protein antigens often contain one or more highly antigenic epitopes (see, e.g., DeLano et al., 2000) that make the search for clones targeting rarer epitopes a time-consuming, laborious and expensive process dominated by large numbers of binders to one or a few dominant epitopes only.
Accordingly, there is a need for the provision of reagents and methods which allow for a more comprehensive interrogation of the full repertoire of epitopes available on antigens of interest, particularly in such cases where there is little or no prior knowledge of the epitopic diversity of such antigens. Such reagents and methods would advantageously thus facilitate the isolation and/or identification of collections of antibodies containing members with epitopic specificities that, are collectively, more reflective of the epitopic diversity of such antigens of interest than other methods in the art.